The von Hippel-Lindau (VHL) disease is a familial cancer syndrome that is dominantly inherited and that predisposes affected individuals to a variety of tumors. The minimum birth incidence of VHL disease is low, being one in 36,000, but the consequence of the disorder is serious in that median actuarial life expectancy is 49 years. In 1993, a gene discovered by positional cloning at human chromosome 3p25-56 was identified as the VHL disease tumor suppressor gene (Latif, F., Tory, K., Gnarra, J., et al. "Identification of the von Hippel-Lindau Disease Tumor Suppressor Gene." Science 260, 1317-1320 (1993)). The cDNA sequence of this gene is the subject of U.S. patent application Ser. No. 08/061,889 U.S. Pat. No. 5,654,138.
In 1994, a related group reported that VHL mutations could be found in a majority of localized and advanced sporadic renal carcinomas. (Gnarra, J. R., Tory, K., Weng, Y. et al. "Mutations of the VHL Tumour Suppressor Gene in Renal Carcinoma". Nature Genetics 7, 85-90 (1994)). This finding substantially widened the scope of clinical interest in the VHL tumor suppressor gene because over 60,000 persons per year in North America alone are diagnosed with kidney growths such as cysts and neoplasms. The detection of VHL gene mutation in biopsies of such growths is therefore diagnostic of a developing tumor, and only those with VHL mutation are thought to be malignant.
Of equal importance, is the finding also reported by Gnarra et al. that of 119 tumors taken from 11 different tissues outside the kidney, not one demonstrated VHL mutation. Such a finding is decisive in determining if a neoplasm is a secondary metastatic tumour of the kidney, or not.
Further information on von Hippel-Lindau disease can be found in the National Cancer Institute, CancerNet database accession number 193300. (http://www.ncc.go.jp/cnet.html).
The cDNA sequence for the von Hippel-Lindau tumor suppressor gene can be obtained from the On-line Mendelian Inheritance in Man database (http://gdbwww.gdb.org/omimdoc/omimtop.htm] GDB ID: G00-120-488; and also in GenBank (http://golgi.harvard.edu/genbank.html) accession number L15409. GenBank also contains sequence disclosed in a recent publication covering the 5' non-translated sequence preceding the initiation codon of the mRNA for the VHL gene. (Kuzmin,I., Duh,F., Latif,F., Geil, L., Zbar,B. and Lerman, M. I. "Identification of the promoter of the human von Hippel Lindau disease tumor suppressor gene" Oncogene 10, 2185-2194 (1995)).
Fragments of various intron sequences can be obtained from the GenBank database G00-361-137; G00-361-147; G00 361-151; G00 375-126; G00 375-133; G00-375-187; G00-375-193; G00-437-732; G00-532-940; G00-532-957; G00-532-965. These sequences have been used as amplification primers for RFLP analysis. However, the intron sequences set out in this application for the purpose set out in this application have not been published.
Methods for identifying VHL tumor suppressor gene mutations have been disclosed in the above noted publications and patent applications. While these methods have had some success in small scale sampling, as demonstrated in the above noted publications, they suffer from practical and theoretical shortcomings. In particular, nucleic acid assays, using cDNA sequences, cannot find mutations in the introns or at the intron/exon boundary of genomic DNA because convenient amplification of such regions requires at least one primer from within the region. Without having at least a small part of the intron amplified, it is impossible to obtain the crucial DNA sequence information by conventional means. Further, mRNA and cDNA are not necessarily the preferred molecules for diagnosis, if genomic DNA is available. This flows from the fact that intact mRNA is often difficult to obtain from patient samples, while genomic DNA sufficient for analysis can usually be obtained even from very small patient samples. Another shortcoming is that one of the most commonly used methods for diagnosing VHL tumor suppressor gene mutations, Southern blot probing, is time consuming and does not lend itself to use in a rapid or low cost diagnostic laboratory.
Currently, no laboratory in the world performs routine VHL mutation diagnostic assays for large numbers of patients. One of the reasons for this is the inability to determine a low cost, high sensitivity and high specificity methodology for routine diagnostic testing of VHL tumor suppressor gene mutations. It is therefore highly desirable to have an improved diagnostic method for the presence or absence of VHL mutation. In particular it would be convenient to develop a diagnostic hierarchical system which could take advantage of the new generation of high speed automated DNA sequencing apparatuses, such as the instruments disclosed in U.S. patent application Ser. No. 08/332,577 now U.S. Pat. No. 5,627,022; Ser. No. 08/353,932, now U.S. Pat. No. 5,507,934; Ser. Nos. 08/332,892; 08/387,272 now U.S. Pat. No. 5,543,018, incorporated herein by reference, which can determine 300 nucleotides (nt) of a sequenced gene in under half and hour. Such a diagnostic hierarchical system is disclosed in the parent application of the instant application, U.S. patent application Ser. No. 08/271,946 now U.S. Pat. No. 5,545,527.
The instant invention provides a low cost, high sensitivity and high specificity methodology for routine diagnostic testing of VHL tumor suppressor gene mutations. It provides intron based primers and combinations of primers which simplify the work of the diagnostic technician. Intron sequences have not previously been used for routine diagnosis of VHL mutations for a number of reasons. First, the sequences themselves have not been known. Second, intron sequences have been expected to show a degree of variability which would make them unreliable for diagnosis. Third, no one had developed an efficient method for using combinations of primers derived from intron sequences.
It is an object of the instant invention to provide a rapid and cost-effective diagnostic hierarchical system for determining the presence or absence of mutation in the VHL gene of a plurality of patients.
It is a further object of the instant invention to provide oligonucleotides from intron regions of the genomic VHL tumor suppressor gene (at human chromosome 3p25-p26) that can be used for the diagnosis of VHL tumor suppressor gene mutation.
It is a further object of the instant invention to provide oligonucleotides from the 5' leader sequence of the genomic VHL tumor suppressor gene (at human chromosome 3p25-p26) that can be used for the diagnosis of VHL tumor suppressor gene mutation.
It is a further object of the instant invention to provide oligonucleotide primers from the regions immediately flanking the intron/exon boundaries of the genomic VHL tumor suppressor gene (at human chromosome 3p25-p26) that can be used for the diagnosis of VHL tumor suppressor gene mutation.
It is a further object of the instant invention to provide kits for the diagnosis of VHL tumor suppressor gene mutation containing these oligonucleotides.